Purification of low molecular weight enzymes

ABSTRACT

For preparation of an enzyme, the enzyme containing solution is passed through an ultrafilter passing only material having a molecular weight lower than 10,000; a soluble form of a bivalent non-toxic cation is added to it, and this cation is precipitated as a carbonate in the enzyme solution; the enzyme is selectively adsorbed to the said carbonate when so precipitated. The carbonate with the adhered enzyme is recovered for example on a filter or by centrifugation and may be washed and/or dried as such, or treated with acid for recovery of the pure enzyme. The metal carbonate complex of the enzyme is particularly suitable for therapeutic purposes because of its high ability and the ready availability of the enzyme upon contact with stomach acid.

United States Patent 1191 Buetow [451 Nov. 27, 1973 PURIFICATION OF LOWMOLECULAR WEIGHT ENZYMES [75] Inventor: Ralph William Buetow, Wausau,

Wis.

[73] Assignee: Johan Bjorksten, Madison, Wis.

[22] Filed: Oct. 16, 1970 [21] Appl. No.: 81,391

[52] US. Cl. 195/66 R, 195/68 [51] Int. Cl C07g 7/02 [58] Field ofSearch 195/62, 66, 68;

[56] References Cited UNITED STATES PATENTS 3,228,876 l/l966 Mahon210/22 FOREIGN PATENTS OR APPLICATIONS 642,653 9/l950 Great Britain195/63 OTHER PUBLICATIONS Ensign, et al., Characterization of a SmallProteolytic Enzyme Which Lyses Bacterial Cell Walls, J. of Bacteriology,Vol. 91, No. 2, 2/l966, (Pp. 524-534) QRIJ8. Michaels, A. S.,Ultrafiltration, Amicon Corporation, Lexington, Mass., 3/1968 BookletNo. 905 (Pp. 12-23).

Dixon, et al., Enzymes, Academic Press Inc., N.Y., 1964, 2nd Ed. (Pp.12, 13 & 29-49). QP. DSeC, 2. Cowman et al., Temperature-DependentAssociation-Dissociation of Streptococcus Lactis, IntracellularProteinase, Biochemical and Biophysical Research Communications, Vol.23, 1966 (Pp. 799-803) QR50lB43.

Primary ExaminerDavid M. Naff Attorney-Johan Bjorksten [5 7] ABSTRACTFor preparation of an enzyme, the enzyme containing solution is passedthrough an ultrafilter passing only material having a molecular weightlower than 10,000; a soluble form of a bivalent non-toxic cation isadded to it, and this cation is precipitated as a carbonate in theenzyme solution; the enzyme is selectively adsorbed to the saidcarbonate when so precipitated. The carbonate with the adhered enzyme isrecovered for example on a filter or by centrifugation and may be washedand/or dried as such, or treated with acid for recovery of the pureenzyme. The metal carbonate complex of the enzyme is particularlysuitable for therapeutic purposes because of its high ability and theready availability of the enzyme upon contact with stomach acid.

11 Claims, 1 Drawing Figure PATENTEDHUY 2 1 ma Enzyme Waste FilirotePURIFICATION OF LOW MOLECULAR WEIGHT ENZYMES PRIOR ART Enzymes havepreviously been purified by adsorption processes including particularlysurface active clays, alumina, kaolin and the like.

Applicant is not aware of any previous work done on enzyme adsorption inwhich metal carbonates have been solubilized with carbon dioxide underpressure, and then precipitated and/or redissolved by changes of Cpressure to effect purification of enzymes by selective adsorption.

Very low molecular enzymes can be extremely unstable in solution, andrequire special methods. This is particularly true of many preparationsof enzymes having a molecular weight below 10,000, such as thosedisclosed and claimed in the co-pending application of Bjorksten andWeyer, Ser. No. 83,523 Filed Oct. 23, 1970 for Enzyme Preparation andProducts.

OBJECTS OF THE INVENTION To find an economically feasible method for thepreparation of substantial quantities of enzymes having molecularweights under 10,000

To produce low molecular enzymes in quantity and at low cost.

To prepare stable, technically and therapeutically useful preparationsof low molecular enzymes.

Further objects will become apparent as the following detaileddescription proceeds.

BRIEF DESCRIPTION OF THE INVENTION In order to concentrate and/orseparate enzyme from a dilute solution, such as a fermentation broth, apress juice, autolysate or macerate of vegetabilic or animalic origin, Ifirst subject this solution to an ultrafiltration through a membrane ormembranes which retain all molecules larger than mol. wt. 10,000. Thisstep is unique in the art, because it has been and is still gener allyassumed that all enzymes have molecular weights well above 10,000, sothat the ordinary person skilled in the art of enzyme purification wouldbe convinced that no activity can pass through such a membrane.

I have found this to be an error, and am basing my process on suchprecleaning, with ultrafiltration removal of all components having amolecular weight higher than 10,000 that is a removal from allpreviously known enzymes.

I then add to the filtrate from this step a non-toxic bivalent cation ina soluble form, or solubilize it after addition, and precipitate it inthe broth, preferably as a carbonate. This nascent bior polyvalent metalcarbonate adsorbs selectively the very low molecular enzymes with whichthis application is concerned.

THE DRAWING Reference is made to the drawing, which is a diagrammaticflow sheet representing a typical embodiment of the invention.

DETAILED DESCRIPTION Referring to the FIGURE, the crude enzyme solutionwhich may be for example a fermentation broth, or an extract,press-juice, macerate or autolysate of vegetabilic or animalic origin isfreed by filtration or centrifu- -gation, 1, from solid suspendedmatter. The resultant liquid is then forced by gas or hydraulic pressurethrough an ultrafilter, 2, which retains everything having a molecularweight larger than about 3'050,000. This is not necessary for theprocess, though it is useful in relieving the load on the followingfilter 3, thus extending its useful life between cleanings.

This filter 3 is an ultrafilter such as Amicon-l0 obtained from theAmicon Corporation, 21 Hartwell Avenue, Lexington, Mass., which retainseverything having a molecular weight above 10,000. This is essential tomy process. It removes all enzymes known to the published or generallyknown prior art, so that according to the status of this art no enzymeshould be present in the filtrate from this filter.

Nonetheless, contrary to all published prior art, I have found thatconsiderable enzyme activity, which may be as high as /a of the totalactivity, is present in the filtrate and can be readily concentratedmore than a thousand fold for example by adsorption to a nascentprecipitate of a selectively enzyme adsorbent solid substance. Since theadsorption is maximal when the particles are very small, an ultrafiltersuch as Amicon, UM-2, which retains everything having a molecular weightabove 1,000 is eminently suitable for this purpose, though other meansfor separating the enzyme adsorbate may be used, such as ordinaryfiltration, centrifugation and any other equivalent process. In theparticular embodiment shown in the illustration, an earth alkalicarbonate (for example calcium, strontium or barium carbonate) is addedto the filtrate in vessel 4, agitated by stirring or shaking means 5,while carbon dioxide is being added from cylinder 6, equipped withpressure regulating means 16. When the carbonate has been dissolvedcompletely, which usually requires 5-60 minutes, the CO pressure isreleased, and a vacuum is applied to remove dissolved carbon dioxide. Ifnecessary, a water soluble organic solvent boiling below about C isadded to accelerate the precipitation of the enzyme-carbonate complex.

This is then gathered on separating means 7, which may be an ultrafilterpreferably cutting off at Mol. Wt. 1,000, or any other filter,centrifuging means or equivalent capable of separating theenzyme-carbonate adsorbate.

This may then be further processed, either by drying, to provide a verystable carbonate complex of an otherwise quite sensitive enzyme, or byelution, for example by addition of acid, to release the enzyme which ifnecessary can be further purified by conventional methods known to priorart and/or lyophilized for more permanent storage. In the former case,conveyor 13 moves the enzyme-carbonate complex to drying means 14, forexample a vacuum drum dryer, and a packaging means 15; while in thelatter case the complex is measured at 9, for example by weighing, orautomatic assay, mixed in vessel 10 with a quantity of acid adequate tobind the metal component and release the enzyme. The acid is measured infrom supplying means 11. The released enzyme is lyophilized at 112.

Having thus explained the principle, I shall further illustrate it withspecific examples.

EXAMPLE I A strain of Bac. cereus, having NRRL number B 3869 was grownin a culture medium composed of 5 grams per liter of Bactopeptone, 3grams per liter yeast extract, 1 gram per liter beta-d-glucose made upto l liter with tap water under the following conditions: 34 Ctemperature, agitation 350 rpm, air flow 2 liters per minute, timeapproximately 72 hours.

The resultant broth was filtered first through an Amicon PM-30 filter toremove molecules larger than 30,000 Mol. Wt., and then through anAmicaon PM-l filter to remove all molecules larger than 10,000 Mol. Wt.This includes removal of all enzymes known to prior art.

To 350 ml of this filtrate was added 0.35 grams of zinc carbonate. Thecomposition was agitated with introduction of carbon dioxide under 80psi pressure, until all of the zinc carbonate was dissolved. Thisrequired 45 minutes. The carbon dioxide pressure was then released, andthe composition placed under a vacuum for /z hour to remove dissolvedcarbon dioxide. Acetone was added in volume equal to that of thecomposition, in order to facilitate separation of the carbonate. Theproduct was left to separate 8 hours. pH was 7.

After standing overnight, the precipitate was removed by ultrafiltrationon an Amicon UM2- membrane. The wet precipitate gave a Congocoll assaynumber of 20, showing that a 100-fold concentration had taken place.

The Congocoll assay employed was a modification of the method of Nelson,et al., A Rapid Method for the Quantitative Assay of ProteolyticEnzymes," Analytical Biochemistry, Vol. 2, pp. 39-44 (1961), and wascarried out as follows: To an appropriate test tube (13 X100 mm size isbest) add 20 mg finely ground (through No. 80 mesh) Congocoll. Add tothe tube 0.1 M tris buffer, pH 7.2, to a volume equal to 1.0 ml minusthe enzyme sample size; i.e., if a 0.1 ml enzyme sample is to be tested,first add 0.9 ml tris buffer. Also, prepare a blank to which the sampleswill be compared by adding 1.0 ml tris buffer to an additional Congocollfilled tube. Immediately place tubes in shaker water bath for 30 minutesat 30 C. After incubation, immediately dilute contents with ml 0.1 Mtris buffer, pH 7.2. Shake and filter through No. 1 Whatman filter paperto remove congocoll. Read collected filtrates at 495 mu balanced againstblank.

The assay numbers at the various steps of the process were as follows:

Original broth (Fermenter II) EXAMPLE 2 A strain of Bac. Cereus, havingNRRL number B 3869 was grown in a culture medium composed 5 grams perliter of BactoPeptone, 3 grams per liter of yeast extract, 1 gram perliter beta-d-glucose made up to 1,000 ml tap water, under the followingconditions: 36 C temperature, agitation 350 rpm, air flow 2%liters/minute, time 65 hours. Cells were removed by Sharplescentrifugation.

The resultant broth was filtered first through an Amicon PM 30 filter toremove the bulk of large molecules above 30,000 Mol. Weight) and thenthrough an Amicon PM 10 filter to remove all molecules larger than10,000 Mol. Wt. This includes removal of all hydrolases known to priorart.

To 500 ml of this filtrate was added 60 milligrams calcium carbonate.The product was then placed under psi CO pressure in a 5 gallonstainless steel container, and agitated under this pressure at 1 C for 1hour. The container was then evacuated, and maintained under vacuum Ahour with continued agitation, for removal of the carbon dioxide,whereupon 500 ml acetone, precooled to 1 C were added. In 3 minutes thesolids had sedimented. About of the supernatant liquid was drawn offclear with a siphon having its end bent upward to minimize bottomsuction. The remainder was filtered on a Whatman No. 2 filter on a 1inch sintered glass funnel, and dried in desiccator. The yield was 780mg of wet precipitate.

3 mg of this precipitate was tested with Congocoll as in Example 1. Theactivity of this quantity was A 1.80. On re-testing 2 months later, theactivity had not changed.

A 10 mg sample of the wet precipitate, having a solids content of 10%,was stored in a sealed vessel in cold room at 1 C. In 6 days theactivity had dropped to 40% of the initial activity. The remainder ofthe preparation was dried in vacuum desiccator over silica gel. Thispreparation was stable. On storage without special precautions at roomtemperature, the activity was unchanged on rechecking after 50 days.

This stable low molecular enzyme preparation is active as such, and isalso active as a stable product slowly releasing enzyme particularly inslightly acidic media. The pH optimum appears to be fairly broad, withgood activity around pH 7. It is particularly useful for the hydrolyticbreakdown of proteinaceous substances where steric hindrances impede theaction of large molecular enzymes.

It appears essential for the present purification method to precipitatethe solid adsorbent in the enzyme containing solution. When solid,finely dispersed zinc carbonate, or calcium carbonate was added to thefermentation broth, and the solubilization and subsequent precipitationsteps omitted, hardly any enzyme activity was adsorbed on the carbonate,from fermentation broth filtrates identical with those of the aboveexamples.

My process has resulted in several hundred fold concentration increaseof enzyme content after removal by the initial ultrafiltration of allenzyme activity due to enzymes having molecular weights above 10,000.

The ultrafilter No. PM-l0 membranes were checked after the enzymefiltration by filtering 0.2% solutions of Cytochrome C (Mol. Wt.12,400), and Lysozyme, the lowest molecular enzymes available to us(Mol. Wt. 14,500). None of these penetrated the ultrafilter membrane,from which we conclude that this had not been damaged but wasfunctioning properly.

While the ultrafiltration step preferably is carried out at thebeginning of the process, as in the above example, I can also carry outthe precipitation step first, elute the adsorbed enzymes, and then carryout the ultrafiltration for selective separations of the very lowmolecular enzymes.

While 1 have disclosed certain specific conditions in the xamples, byway of illustration and not of limitation, 1 have found that theinvention is capable of considerable variation.

The most essential part of the invention resides in the discovery thatenzymes can be separated by a method which as an essential stepcomprises ultrafiltration which will effectively'remove all hydrolyticenzymes previously known to exist, as no hydrolytic enzyme having amolecular weight less than 10,000' has even before been reported in theliterature.

This ultrafiltration step may be followed by adsorption of the enzyme oractive enzyme fragment I have found to be still present, on a solidmedium which is precipitated out in the presence of the very lowmolecular enzyme. Particularly suitable solid adsorbants are the earthalkali carbonates, magnesium carbonate and aluminum carbonates, whichall are effective adsorbents when dissolved under carbon dioxidepressures of 2 to 2,000 psi, and reprecipitated by removal of the excesscarbon dioxide. These adsorbents can be used to advantage in percentagesfrom 0.01% to by weight of the total mix. The resultant carbonate-enzymecomplex can be dried for storage or the enzyme can be liberated byelution, and lyophilized in accordance with practices well known in theart. These complexes appear indefinitely stable on storage dry at roomtemperatures.

The elution may be carried out for example by resolubilizing thecarbonate with CO pressure, or by cautious addition of dilute acidsolution, for example by placing the enzyme adsorbate in an atmosphereinto which an acid such as for example maleic, acetic, hydrofluoric orhydrochloric is slowly evaporating: or by having the dilute acid passthrough the adsorbate in a column. However, in these elution steps wehave invariably lost some activity, and where compatible with the enduse we therefore prefer to use the enzyme in the adsorbed state,substantially as obtained from the concentration process of example 2.The resultant product then contains a hydrolase having molecular weightbetween 1,000 and 10,000 and preferably between 3,000 and 7,500, and acarbonate of a metal selected from te group consisting of earth alkalimetals, zinc, magnesium and aluminum, said hydrolase preparation beingstable when dry and being active at pH 7. For therapeutic purposes Iprefer to employ a hydrolase being adsorbed to a non-toxic metalcarbonate, such as a carbonate of calcium, magnesium or aluminum.

The pure enzyme can be liberated by redissolving the metal carbonateadsorbate in water under CO pressure, then passing the resultantliquidthrough an ultrafilter with cut-off at 1,000 to 3,000 mol. weight, sothat the dissolved carbonate passes through, while the pure enzymeremains on the filter.

The enzymes thus liberated have molecular weights within the range 1,00010,000; more often 3,000 7,500. Molecular weight of 6,400 has been foundseveral times in protease concentrates made from fermentation broths ofdifferent microorganisms, for example NRRL B-3867; 8-3868; 8-3871;B-3872; 13-3880. These are highly active on casein as well as on theCongocoll assay methods. The above organisms are available to the publicfrom the type collection of the U.S. Department of Agriculture, NorthernUtilization Research & Development Division, Peoria, 1]].

While most of the work on which this application is based has been donewith proteases, and found to apply to these quite generally, it may alsobe applicable to cellulases, amylases, lipases, catalase, peroxidase,peptidases, particularly in the presence of proteases, since these tendto excise some active groups in the course of attack on other enzymes.It is believed to apply to virtually all enzymes because they all haveactive groups connected with large aggregates, which can be pared downby proteases without destroying the active configurations.

Regardless of whether or not this is the actual mode of action, it isclear that there is utility as well as novelty in the present methodwhich makes available in a practical way active and stable enzymepreparations having very much smaller molecules than known by prior art,and therefore capable of greater penetration into sterically difficultyaccessible sites or structures. Fields of utility include, but are notlimited to, for example, hydrolysis of waste products, stain removal,and therapeutic uses.

Having thus disclosed my invention, I claim.

1. The method for concentrating a protease, which comprises the steps ofpassing a crude protease solution through at least one ultrafiltermembrane retaining substances of molecular weight exceeding 10,000 toproduce a filtrate containing said protease; adsorbing the protease insaid filtrate by adding a carbonate of the group selected from earthalkali metals, magnesium and aluminum, in quantity of 0.01 to 10% on thetotal quantity in process; solubilizing by agitating under a elevated COpressure; precipitating after removal of carbon dioxide bydepressurizing; separating and drying the resultant carbonate proteasecomplex.

2. The method of claim 1, in which the carbonate used is calciumcarbonate and the carbon dioxide pressure used for solubilizing it isbetween 2 and 2,000 psi.

3. The method of claim 1, in which the carbonate is magnesium carbonateand the carbon dioxide pressure used for solubilizing it is between 2psi and 2,000 psi.

4. The method of claim 1, in which the crude protease solution is firstfiltered through an ultrafilter retaining material having a molecularweight range of 30,000 to 50,000; then through an ultrafilter retainingsubstances of molecular weight exceeding 10,000, whereafter the filtratepassing through both of these filters is exposed to an in situprecipitated enzyme adsorbent selected from the class consisting ofcalcium carbonate, zinc carbonate, magnesium carbonate and aluminumcarbonate, separating washing and drying the resultantprotease-carbonate complex.

5. The method of claim 2, in which the aforesaid metal carbonates areadded in a proportion of 0.01 to 10% by weight on the crude proteasecontaining solution, dissolved by agitation under CO pressure in therange of 2 2,000 psi, and reprecipitated by removal of carbon dioxide.

6. The method of claim 3, in which a water miscible organic solventhaving a boiling point below C is added to facilitate the separation ofthe nascent metal carbonate.

7. The method of claim 3 in which the said carbonate is calciumcarbonate, and the said CO pressure is 2 2,000 psi, the said CO removalis by application of vacuum for 10-30 minutes and the said solvent isacctone in quantity equal to the volume of the water present.

8. The method for concentrating a protease which comprises the steps ofpassing a fermentation broth of a microorganism through an ultrafiltermembrane retaining substances of molecular weight exceeding 10,000 toproduce a filtrate containing said protease, adding to said filtrate acarbonate selected from the group consisting of earth alkali-metals,magnesiumand aluminum, solubilizing said carbonate by carbon 10. Themethod of claim 9, the said carbonate being calcium carbonate.

11. The method of claim 9, a water miscible organic solvent being addedto facilitate the precipitation of 9. The method of claim 8 in whichsaid organism is said carbonate.

a strain of Bacillus cereus.

2. The method of claim 1, in which the carbonate used is calciumcarbonate and the carbon dioxide pressure used for solubilizing it isbetween 2 and 2,000 psi.
 3. The method of claim 1, in which thecarbonate is magnesium carbonate and the carbon dioxide pressure usedfor solubilizing it is between 2 psi and 2,000 psi.
 4. The method ofclaim 1, in which the crude protease solution is first filtered throughan ultrafilter retaining material having a molecular weight range of30,000 to 50,000; then through an ultrafilter retaining substances ofmolecular weight exceeding 10,000, whereafter the filtrate passingthrough both of these filters is exposed to an in situ precipitatedenzyme adsorbent selected from the class consisting of calciumcarbonate, zinc carbonate, magnesium carbonate and aluminum carbonate,separating washing and drying the resultant protease-carbonate complex.5. The method of claim 2, in which the aforesaid metal carbonates areadded in a proportion of 0.01 to 10% by weight on the crude proteasecontaining solution, dissolved by agitation under CO2 pressure in therange of 2 - 2,000 psi, and reprecipitated by removal of carbon dioxide.6. The method of claim 3, in which a water miscible organic solventhaving a boiling point below 180* C is added to facilitate theseparation of the nascent metal carbonate.
 7. The method of claim 3 inwhich the said carbonate is calcium carbonate, and the said CO2 pressureis 2 - 2,000 psi, the said CO2 removal is by application of vacuum for10-30 minutes and the said solvent is acetone in quantity equal to thevolume of the water present.
 8. The method for concentrating a proteasewhich comprises the steps of passing a fermentation broth of amicroorganism through an ultrafilter membrane retaining substances ofmolecular weight exceeding 10,000 to produce a filtrate containing saidprotease, adding to said filtrate a carbonate selected from the groupconsisting of earth alkali-metals, magnesium- and aluminum, solubilizingsaid carbonate by carbon dioxide pressure, precipitating said dissolvedcarbonate in the presence of said protease by withdrawal of said carbondioxide pressure and separating the resultant protease carbonatecomplex.
 9. The method of claim 8 in which said organism is a strain ofBacillus cereus.
 10. The method of claim 9, the said carbonate beingcalcium carbonate.
 11. The method of claim 9, a water miscible organicsolvent being added to facilitate the precipitation of said carbonate.